Histone Modifications / Antibodies
Quantifying histone modifications using mass spectrometry (Prot 51)
A variety of different posttranslational modifications (PTMs) on histone proteins display an important layer of chromatin regulation. PTMs mostly occur on the N-terminal tails of histones that are protruding out of the nucleosome, with histones H3 and H4 featuring more described PTMs than H2A and H2B (Figure 1). Among the set of known histone PTMs, methylation and acetylation are the most abundant. In order to understand more about chromatin regulation, it is crucial to analyze histone modifications in a quantitative way.
Different technical approaches are available to determine the amount and location of these histone modifications, with antibody-based techniques and mass spectrometry being the most popular. We will describe a procedure to isolate histones, prepare them for MS analysis and interpret the results (Peters et al, 2003). [...]
Munich Center of Integrated Protein Science (CIPSM) and Adolf-Butenandt-Institut, Schillerstr. 44, 80336 München, Germany
Corresponding author: Ignasi Forne, Teresa Barth
TSA Treatment of Mammalian Cells (Prot 9)
Prolonged treatment of proliferating mammalian cells with low doses of a histone deacetylase inhibitor, trichostatin A, specifically affects pericentric heterochromatin. Relocation of these regions towards the nuclear periphery and loss of HP1 proteins ensue after several divisions. Meanwhile, DNA methylation state and core centromeric markers are maintained. Subsequent defects in centromeric function arise in mitosis. Remarkably, removal of the drug rapidly reverses all these changes.
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